Mitochondria-associated membrane protein PACS2 maintains right cardiac function in hypobaric hypoxia

Summary Hypobaric hypoxia (HH) is the primary challenge at highland. Prolonged HH exposure impairs right cardiac function. Mitochondria-associated membrane (MAM) plays a principal role in regulating mitochondrial function under hypoxia, but the mechanism was unclear. In this study, proteomics analysis identified that PACS2, a key protein in MAM, and mitophagy were downregulated in HH. Metabolomics analysis indicated suppression of glucose and fatty acids aerobic oxidation in HH conditions. Cardiomyocyte Pacs2 deficiency disrupted MAM formation and endoplasmic reticulum (ER)-mitochondria calcium flux, further inhibiting mitophagy and energy metabolism in HH. Pacs2 overexpression reversed these effects. Cardiac-specific knockout of Pacs2 exacerbated mitophagy inhibition, cardiomyocyte injury, and right cardiac dysfunction induced by HH. Conditional knock-in of Pacs2 recovered HH-induced right cardiac impairment. Thus, PACS2 is essential for protecting cardiomyocytes through ER-mitochondria calcium flux, mitophagy, and mitochondrial energy metabolism. Our work provides insight into the mechanism of HH-induced cardiomyocyte injury and potential targets for maintaining the right cardiac function at the highland.


INTRODUCTION
High-altitude areas cover much of the total geographical area worldwide, and human exposure to high altitudes is increasing for various reasons. Hypobaric hypoxia (HH) caused by exposure to increasing altitude is the main physiological challenge in such conditions and has long been recognized as a cause of cardiac stress. Acute exposure to high altitudes induces an increase in the right ventricular (RV) afterload, leading to the alteration of the RV filling patterns. 1 Prolonged exposure to high altitude further results in chronic remodeling of the cardiac structure and function, ultimately leading to right heart failure. 2,3 Among these cardiac adaptive and/or pathological alterations, cardiomyocyte responses, particularly in intracellular homeostasis maintenance during hypoxia, are the critical molecular basis that determines the adaptive cardiac outcomes. 4,5 Cardiomyocytes consume the majority of oxygen in the mitochondria as an electron donor for oxidative phosphorylation (OXPHOS). [6][7][8] Thus, mitochondria are highly sensitive to decreases in oxygen levels in cardiomyocytes. Hypoxia increases oxidative stress and mitochondrial DNA mutation and causes mitochondrial dysfunction. 9 Hypoxia also suppresses mitochondrial OXPHOS and leads to the accumulation of aerobic metabolic substrates and anaerobic metabolites, which result in cardiomyocyte injury and cardiac dysfunction. 10 These damaged mitochondria and excessive metabolic substrates could be removed via mitophagy. 11,12 However, mitophagy is suppressed in some cardiac diseases, such as diabetic cardiomyopathy and ischemic cardiomyopathy, 13 which leads to mitochondrial dyshomeostasis and cardiac dysfunction. Therefore, an appropriate level of mitophagy serves as a protective mechanism to maintain the mitochondrial function in response to cardiac stress. 14 In recent years, several mitochondrial membrane receptors containing the LC3-interacting region (LIR) motif have been found to mediate mitophagosome formation under acute hypoxic conditions. 11, 15 However, there is a paucity of information regarding cardiomyocyte mitophagy during chronic HH exposure, and the precise mechanism has not been fully elucidated.
In this study, we established HH conditions to closely simulate high-altitude exposure and focused on PACS2-mediated mitophagy and mitochondrial energy metabolism, regarding calcium flux across the MAM as the core mechanism. This study provides insights into the cardiomyocyte response to HH conditions. We also interpreted the mechanism underlying high-altitude-induced right cardiac dysfunction.

RESULTS
Hypobaric hypoxia exposure induces the downregulation of PACS2 and affects mitophagy and mitochondrial energy metabolism in the right myocardium C57BL/6J mice were assigned to an HH chamber for 6 weeks for the simulation of high-altitude conditions ( Figure 1A). We first performed proteomics and metabolomics analyses accordingly in the right myocardium of mice that had been allowed to develop hypoxia-induced pulmonary hypertension after the 6-week chronic HH exposure. The different candidates were defined using a criterion of R1.2 log 2 fold change and a significant difference between the groups. In the proteomics analysis, we identified 217 downregulated proteins and 82 upregulated proteins in the right myocardium of HH-exposed mice when compared with the respective levels in the normobaric normoxia (NN) counterparts ( Figure 1B and Table S1). In the metabolomics analysis, we identified that 53 endogenous metabolites increased and 22 decreased (Table S2). Hierarchical clustering analysis of metabolomics indicated markedly altered cardiac metabolic pathways under HH exposure ( Figure 1C). Among the differentially expressed proteins, we identified PACS2 as being significantly downregulated by 3.16-fold ( Figure 1D). In addition to PACS2, MAP1LC3A, MAP1LC3B, autophagy-related 16-like 1, and sequestosome 1 (SQSTM1/p62) were remarkably downregulated; these were involved in phagophore formation and mitophagy induction ( Figure 1E). To gain insight into the possible biological effect of HH exposure, we subjected proteins that were downor upregulated to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway enrichment analysis. Glycolysis, the HIF-1 signaling pathway, and focal adhesion were upregulated during HH, while OXPHOS, the citrate cycle, fatty acid beta-oxidation, and mitophagy were downregulated ( Figures 1F and 1G). The above-mentioned results indicated that both mitophagy and mitochondrial energy metabolism were impaired in the right myocardium of the mice owing to HH exposure.
Cardiac Pacs2 ablation exacerbated the right cardiac dysfunction and structure impairment induced via hypobaric hypoxia exposure To determine the role of PACS2 in maintaining right cardiac function and structure, we generated cardiomyocyte-specific Pacs2 cKO (Pacs2 flox/flox /Cre aMHC+/À ) mouse models (Figure 2A) 2M); however, the heart weight was not further changed in Pacs2 cKO mice ( Figure 2K). hematoxylin-eosin (HE) staining further revealed increased RV chamber thickness, decreased RV chamber size, and disordered arrangement of RV myocardium caused by cardiac Pacs2 ablation ( Figure 2N). Masson's trichrome staining showed significant collagen deposition in the right myocardial interstitial space after HH exposure ( Figures 2O and 2P). The mean cross-sectional area (CSA) of the RV cardiomyocytes in the HH group was significantly larger than that in the NN group ( Figures 2Q and 2R). Such substantial cardiac remodeling caused by HH was significantly more serious in the hearts of Pacs2 cKO mice. The cardiomyocyte injury was also evidenced by plasma markers (B-type natriuretic peptide [BNP], TnI, and CK-MB), which were much higher in Pacs2 cKO mice than in the remaining two groups ( Figures 2S-2U). Our results indicated that HH-induced right cardiac impairment phenotype became more noticeable following Pacs2 ablation.
To verify the separate role of Pacs2 ablation, we evaluated the right cardiac function under NN conditions. Compared with littermate controls, Pacs2 cKO mice showed a normal mPAP (Figures S1C and S1F), which  iScience Article was accompanied by impaired right cardiac function, as revealed by the lower RV FAC (Figures S1A and S1D) and an increased Tei index (Figures S1B and S1E). Pacs2 cKO mice in NN exhibited significantly increased myocardial disorder and fibrosis ( Figures S2A-S2C). Additionally, cardiomyocyte injury was also evident in NN conditions in the presence of Pacs2 cKO ( Figure S2D-S2H). In general, Pacs2 ablation exacerbated the cardiomyocyte injury and right cardiac dysfunction; however, it did not act on the RV afterload during HH exposure.
Cardiac Pacs2 ablation exacerbated mitochondria-associated membrane disruption and mitophagy reduction induced via hypobaric hypoxia To determine how PACS2 responds to HH, we compared the subcellular localization of MAM-associated proteins in isolated right myocardium under HH or NN conditions. As shown in Figure 3A, different fractions were identified with the following organelle markers: FACL4, VDAC1, MFN2, FIS1, CNX, and TOMM20. The level of PACS2 in MAM significantly decreased in the HH group compared with its levels in the NN counterparts, although a small amount of PACS2 could also be found in the cytosol. However, the levels of other MAM-related proteins were not noticeably altered in MAM fractions isolated from the HH group. Notably, the Pacs2 gene expression was also significantly reduced in HH hearts when compared with NN controls ( Figure 3B). To assess whether PACS2 affects MAM integrity, we examined the ER-mitochondrial contacts in Pacs2 cKO mice myocardium. As illustrated by the TEM images ( Figures 3C and 3D), the proportion of ER in close contact with mitochondria relative to the total ER content was lower in the HH group than in the NN group and further decreased in the Pacs2 cKO mice. Consistent with the TEM images, immunofluorescence analysis clearly showed a lower level of co-localization of the ER with mitochondria in the Pacs2 cKO mice than in the remaining groups ( Figures 3E and 3F).
Further, we determined whether PACS2 alteration affected mitophagy. Decreased mitophagy markers confirmed impaired mitophagy induced by HH exposure in the right myocardium. The blotting results indicated that the level of MAP1LC3B-II was lower in the Pacs2 cKO mice than in controls during HH conditions ( Figures 3G and 3H). Moreover, immunostaining analysis revealed that Pacs2 deletion further reduced the co-localization of MAP1LC3B puncta and mitochondria induced by HH exposure ( Figures 3I and 3J). Combined, cardiac Pacs2 ablation exacerbated MAM disruption and mitophagy reduction induced by HH.

Hypobaric hypoxia reduced mitochondria-associated membrane formation and mitophagy in vitro
To explore the effect of HH on the MAM structure and biological function, we also measured the levels of MAM-related proteins in H9C2 cardiomyocytes exposed to simulated HH in vitro. As depicted in Figure 4A, in line with the in vivo results above, the expression of PACS2 in the MAM from HH-treated cells was lower than that in the MAM from NN-treated cells. Consistently, confocal imaging showed a decreased association between the ER and mitochondria in simulated HH-treated cardiomyocytes compared with that in the NN cells ( Figures 4B and 4C). TEM imaging showed swollen mitochondria and fewer mitochondria adjacent to the ER after HH exposure ( Figure 4D). With respect to the mitochondria-associated ER membranes, we also found a decrease in the ratio of close MAM contacts and relative length ( Figure 4E). These data suggested that HH decreases the MAM junction structure in cardiomyocytes. Furthermore, we evaluated mitophagy levels in H9C2 cardiomyocytes. We found decreased MAP1LC3B-II transfer ( Figures 4F and 4G) and co-localization with mitochondria after HH exposure ( Figures 4H and 4I). To further verify the impaired mitophagy, we transfected the pH-dependent mitochondrial protein Keima into the cardiomyocytes; this can shift from green to red as mitochondria are delivered to lysosomes. Laser scanning confocal microscope (LSCM) monitoring showed that HH induced a markedly decreased mitophagy index in the cardiomyocytes ( Figures 4J and 4K), indicating that HH decreased the number of mitophagosomes and impaired the mitophagy flux. The above results in the H9C2 cell lines as well show that HH reduces MAM formation and mitophagy. iScience Article hypobaric hypoxia reduced endoplasmic reticulum-mitochondria calcium flux and mitochondrial oxidative phosphorylation in vitro PACS2 was reported to maintain the junction of the MAM and regulate mitochondrial calcium flux. 22 In this study, we found that [Ca 2+ ] m in the H9C2 cardiomyocytes after HH treatment was markedly lower than in the NN group ( Figures S4A and S4B). To determine the origin of mitochondrial calcium, we incubated H9C2 cardiomyocytes under HH or NN with a cytoplasmic Ca 2+ chelator, BAPTA-AM (10 mM), in calcium-free Hanks' balanced salt solution (HBSS) for 10 min. Mitochondrial calcium was labeled using a Rhod2-AM probe, and the cells were observed and measured under LSCM. TG-(a calcium pump inhibitor, Figures 5A and 5B) and ATP-(an indirect IP3R agonist, Figures 5C and 5D) elicited ER-mitochondria calcium flux was lower in HH-treated cells than in NN-treated cells. Inositol trisphosphate receptors (IP3R) are important ER calcium-release channels. 27 Therefore, we added 2-APB, which blocked the release of calcium from IP3R. Comparable mitochondrial calcium to HH exposure was observed when treated with 2-APB ( Figures 5E and 5F), suggesting that IP3R is required for maintaining the physiological mitochondrial calcium levels under NN conditions. Next, we detected the effect of HH on the expression of two calcium transporters majoring in conveying calcium flux in the contact sites of ER and mitochondria, IP3R, and the mitochondrial calcium uniporter (MCU). As shown in Figures S3A and S3B, the expression of both two calcium channel proteins did not significantly change between NN and HH conditions. It is not the calcium transporters that limit the calcium flux across MAM, but the disruption of MAM contact was more likely to affect the calcium flux.
MAM formation and ER-mitochondrial calcium flux is essential for mitochondrial energy metabolism. Thus, we evaluated the effects of HH on cardiomyocyte mitochondrial energy metabolism using a Seahorse XF analyzer to measure mitochondrial respiration and glycolytic flux. We found that cardiomyocytes exhibited significant decreases in basal and maximal cellular oxygen consumption rate (OCR) in response to HH. ATP production and spare respiration capacity were also significantly lower after HH exposure (Figures 5G and 5H). ECAR results indicated an increase in glycolysis and glycolytic capacity owing to insufficient oxygen ( Figures 5I and 5J). In addition, OCR measurement with a medium containing BSA-conjugated palmitic acid significantly decreased after HH exposure ( Figures 5K and 5L). The above-mentioned results show that after HH exposure, the cardiomyocytes displayed metabolic reprogramming, represented by the restriction of FAO-related OXPHOS and a tendency to rely more on anaerobic than aerobic glycolysis for adapting to the HH condition. Since HH caused a decline in ER-mitochondria calcium flux, one mechanism that potentially accounts for the metabolic shift may be associated with the regulation of mitochondrial calcium.
Endoplasmic reticulum-mitochondria calcium flux is involved in PACS2-mediated mitophagy and mitochondrial energy metabolism We next determined the contributions of ER-mitochondria calcium in PACS2-induced mitophagy and mitochondrial energy metabolism. We obtained cardiomyocytes with stable overexpression of Pacs2 through lentiviral vectors (LVVs) infection ( Figures 6A and 6B). ER-mitochondria contacts increased in cells where Pacs2 was overexpressed ( Figures 6C and 6D). We found reversed [Ca 2+ ] m in LVVs-infected cardiomyocytes ( Figures S4A and S4B). Similar results were observed with TG ( Figures 6E and 6F) or ATP (Figures 6G and iScience Article 6H) treatment in Pacs2-overexpressed cultured cells in the dynamics of mitochondrial calcium flux, indicating a source of calcium flux released from the ER. The regulation of calcium flux between the ER and mitochondria via IP3R is a major function of the MAM. 28 As depicted in Figures 6I-6L, the restored calcium levels caused by the overexpression of Pacs2 were partly blocked by 2-APB, suggesting that Pacs2 overexpression promoted ER calcium release in the MAM through IP3R. Additionally, higher MAP1LC3B-II levels were observed after LVV-overexpression of Pacs2 ( Figures 6M and 6N), which could also be blocked by 2-APB ( Figures 6O and 6P), suggesting that PACS2-mediated ER-mitochondria calcium flux was required for mitophagy.
With the supplementation of PACS2, more MAP1LC3B puncta co-localized with mitochondria ( Figures 7A  and 7B) and an increased mitophagy index were observed in HH conditions ( Figures 7C and 7D). These data showed that PACS2 restored impaired mitophagy through enhanced ER-mitochondria calcium flux.
To investigate whether ER-mitochondria calcium flux was also involved in PACS2-mediated mitochondrial energy metabolism alteration, we compared real-time changes in OCR and ECAR in the H9C2 cardiomyocytes with or without overexpression of PACS2 under HH treatment. With PACS2 supplementation, the decreased basal respiration, ATP production, and maximal respiration ( Figures 7E and 7F) and increased basal and maximal ECAR ( Figures 7G and 7H) induced by HH were significantly reversed. The recovery in mitochondrial respiration was also blocked by 2-APB treatment ( Figures 7G and 7H). To extend the hypothesis that PACS2 helped recover OCR, which is supported by FAO, we further measured OCR in a medium containing palmitate-BSA as an exogenous FAO substrate. Notably, the cardiomyocytes showed a reversed OCR after the supplementation of PACS2 when compared with an empty vector control ( Figures 7I and 7J), which was significantly blocked on adding 2-APB. This indicates that PACS2 enabled HH-treated cardiomyocytes to switch from glycolysis to an increased reliance on FAO for ATP production. This metabolic reprogramming at least partly depends on the calcium flux across MAM. Together, these data suggested that ER-mitochondria calcium flux was essential for PACS2-mediated mitophagy maintenance and mitochondrial energy metabolism after HH exposure.
Cardiac Pacs2 knock-in alleviated hypobaric hypoxia-induced right cardiac dysfunction LVVs-overexpression of Pacs2 significantly reversed MAM formation, mitophagy, and mitochondrial energy metabolism in cardiomyocytes in vitro. To verify the contributions of PACS2 in maintaining RV function during HH exposure in vivo, we generated cardiomyocyte-specific Pacs2 knock-in mouse models (Pacs2 cKI; Figure 8A). Histological analysis of the hearts from the Pacs2 cKI mice showed significantly decreased right cardiac hypertrophy ( Figure 8B), cardiac fibrosis area ( Figures 8C and 8D), and cardiomyocytes CSA ( Figures 8E and 8F) with HH exposure. In addition, the Pacs2 cKI mice had lower plasma levels of BNP, TnI, and CK-MB ( Figures 8G-8I) than their littermate controls, indicating that PACS2 supplementation reduced the HH-induced myocardial damage. Compared with their littermate controls, the Pacs2 cKI mice exhibited a higher RV FAC (Figures 8J and 8M) and lower Tei index ( Figures 8K and 8N). As expected, Pacs2 overexpression did not significantly alter mPAP, max dP/dt, and RV VTI (Figures 8O-8Q) during HH exposure. Pacs2 cKI did also fail to completely reverse the impaired cardiac function to the baseline level as the NN group, as shown in Figure S5. Additionally, in the NN condition, Pacs2 cKI had little effect on the normal right cardiac function, representing by no significant difference in FAC, Tei index, mPAP, et al. when compared with their littermate controls. The survival of mice without HH exposure were higher than those in HH exposure groups. Compared with WT mice without HH exposure, survival rate was significantly increased in the Pacs2 cKI group while decreased in the Pacs2 cKO group ( Figure 8R). These data suggested that conditional Pacs2 cKI reduced cardiomyocyte injury and partially recovered RV cardiac function after HH exposure without significantly influencing the RV afterload. With the progressive right cardiac injury caused by HH, the left cardiac function in mice has also been affected significantly. We observed a significant increase in the left ventricular dimensions (end-systolic diameter and end-diastolic diameter) and a significant decrease in the systolic function (left ventricular ejection fraction and left ventricular fractional shortening) in the mice with HH exposure compared to the NN group ( Figures S6A-S6D). Moreover, the effect of PACS2 genetic manipulation appeared to be significant in altering the left cardiac function, as shown by aggravated left cardiac dysfunction caused by Pacs2 cKO and alleviated HH-induced left cardiac dysfunction in Pacs2 cKI mice (Figures S6A-S6D). iScience Article DISCUSSION This is the first study to reveal the underlying mechanism of PACS2 in HH-mediated cardiomyocyte injury and right cardiac dysfunction. The core process was the downregulated PACS2 localized in the MAM after HH exposure. PACS2 reduction further suppressed MAM formation and resulted in decreased calcium flux from the ER to the mitochondria via the IP3R calcium channel. The reduced mitochondrial calcium influx further inhibited mitophagy and mitochondrial energy metabolism, inducing cardiomyocyte injury and right cardiac dysfunction. Moreover, cardiomyocytespecific knock-in of Pacs2 reversed right cardiac dysfunction and RV fibrosis. Of note, neither conditional cKO nor cKI of the Pacs2 in cardiomyocyte influenced the RV afterload, highlighting an independent role of PACS2-directed cardiomyocyte responses in maintaining right cardiac function. Thus, our results provided potential therapeutic targets for high-altitude-induced right cardiac impairment. iScience Article Sufficient oxygen supply is the most essential condition for the survival and function of cardiomyocytes. Hypoxia induces pulmonary vasoconstriction and increases pulmonary vascular resistance. Prolonged hypoxia further results in right cardiac function impairment and even right heart failure. 29 Thus, in this study, we focused on the right rather than on the left cardiac function. Hypoxia is usually generated by normobaric hypoxia (NH) or HH in the experiments. NH lowers the partial pressure of inspired oxygen (PiO 2 ) by reducing the fraction of inspired oxygen by adding exogenous nitrogen without altering the barometric pressure. Conversely, HH lowers the PiO 2 by reducing the barometric pressure. Previous studies have suggested that NH and HH induced similar cardiac adaptations over a short duration, although lower SpO 2 and worse right cardiac function emerged during long-term exposure. 30,31 Thus, more complicated mechanisms may exist in HH than in NH, including intravascular bubble formation, increased alveolar dead space, altered fluid permeability, and a mismatch in ventilation and perfusion. 32 Our study established a long-term HH exposure model to simulate real cardiac function alteration and cardiomyocyte response in high-altitude environments. Previous studies on cardiomyocyte injury caused by hypoxia mainly focused on the increase in oxygen free radicals and anaerobic metabolites, eventually leading to cardiomyocyte apoptosis, myocardial fibrosis, and irreversible cardiac remodeling. 33 Our study indicated that both mitophagy and mitochondrial energy metabolism were involved in cardiomyocytesurvival under HH conditions. Our results revealed a mechanism that results in right cardiac dysfunction at high altitudes.
Previous studies reported that PACS2 is closely associated with the onset and progression of tumors, such as colorectal and liver cancer. 34,35 Owing to their infinite proliferation ability, tumor cells survive in relative hypoxic conditions. Therefore, it is essential to understand the biological function of PACS2 in regulating cell fate in response to hypoxic conditions. Accordingly, after HH exposure, PACS2 expression was found to be markedly reduced in the MAM, although with a moderate increase in PACS2 in the cytosol both in vivo and in vitro (Figures 4A and 5A). This implied a dynamics translocation from the MAM to the cytosol, which may partly explain the decreased PACS2 in the MAM. The free form of PACS2 in the cytosol contributed to nuclear gene expression and membrane trafficking rather than calcium flux, thus exhibiting the suppression of downstream mitophagy and energy metabolism. Besides PACS2, recent studies have found that the FUN14 domain containing 1 (FUNDC1), a new protein in the MAM, is responsible for the release of calcium from the ER to the mitochondria and mitophagy induction in mouse cardiomyocytes. 36,37 Notably, our results revealed that PACS2 served as a mitophagic regulator in the MAM and modulated calcium release from ER to mitochondria in cardiomyocytes. However, the precise molecular mechanism of how PACS2 cooperates with FUNDC1 to regulate calcium flux, mitophagy, and cardiac function remains unknown. We considered that a few special proteins in the MAM may interact to form a protein complex or ''protein machine'' and involve themselves in the above-mentioned process. PACS2 may be the key protein and serves as a scaffold to sponge other proteins.
Mitophagy is essential for mitochondrial homeostasis and quality control in cardiomyocytes. 38 During hypoxia, mitophagy is the sole mechanism through which cardiomyocytes eliminate superfluous or damaged mitochondria. 14 However, the mechanisms underlying mitophagy remain largely unknown. Previous studies on mitophagy have focused on several protein receptors on the mitochondrial membrane, including BCL2 interacting protein 3 (BNIP3), BNIP3-like, and FUNDC1. Most of them have a classic LIR motif to directly bind MAP1LC3B for mitophagy activation. 39 In this study, we found a new protein in the MAM without this classic LIR motif, although it was closely associated with HH-mediated mitophagy. PACS2 did not directly link to autophagy-associated proteins, such as ATG5, ATG7, Beclin1, and MAP1LC3B; however, it acted as a calcium channel to promote calcium influx into the mitochondria. 22 Usually, intracellular calcium is considered an activator of autophagy. [40][41][42] To date, the role of calcium signaling in autophagy regulation is highly controversial. Most studies considered that calcium works as an autophagy activator because calcium mobilizing agents and calcium ionophores promote autophagy by elevating intracellular calcium concentration. 43 In this study, we detected that PACS2-mediated calcium influx was required for HH-induced mitophagy in cardiomyocytes, which iScience Article further verified the effect of intracellular calcium on mitophagy regulation. However, the mechanism through which mitochondrial calcium is involved in mitophagy activation requires further exploration. Mitochondrial calcium uptake occurs mostly through MAM, which closely contacts with the ER and renders a micro-domain with a sufficiently high calcium concentration. 44 A recent study reported that mitochondrial calcium influx inhibition decreased ATP production, enhanced mitophagy, and provided cardioprotection in cardiac failure. 45 Conversely, we found that HH decreased ER-mitochondria calcium flux and iScience Article calcium-mediated mitophagy. Similar to our results, Bö ckler and Zou demonstrated that ER-mitochondria contact and calcium flux across the MAM were required for autophagic removal of mitochondria since artificially tethering ER and mitochondria rescued mitophagy defects. 18,33 Besides mediating calcium flux, the ER-mitochondria encounter structure may also supply the growing phagophore with lipids synthesized in the ER, which then enclose the impaired mitochondria to form a mitophagosome. Hence, ER-mitochondria-mediated calcium flux is required for mitophagy induction.
In addition to the above-mentioned role of calcium in mitophagy, mounting evidence suggests that calcium also dynamically regulates the aerobic energy metabolism by stimulating mitochondrial OXPHOS. 46,47 In highly energy-consuming tissues, such as the heart, OXPHOS in the mitochondria provides a major source of cellular ATP through the oxidation of substrates, including fatty acids, glucose, and ketones. 48 We found that to adapt to the HH condition, cardiomyocytes mainly rely on the glycolytic iScience Article pathway rather than the OXPHOS pathway. As a critical signaling molecule in mitochondrial energy conversion, sufficient mitochondrial calcium concentration is required to activate mitochondrial dehydrogenases, including the pyruvate dehydrogenase complex (PDHC), NADH-isocitrate dehydrogenase (ICDH), and a-ketoglutarate dehydrogenase (a-KGDH). 49,50 Other components within the energy-producing pathways besides NADH generation, such as downstream ATPase and the cytochrome chain, were also significantly stimulated by calcium. 51,52 Current studies supported the proposed physiological metabolic role of calcium entry into the mitochondria matrix through the mitochondrial calcium uniporter (MCU) complex. 53 Together with some recent reports, we indicated that the IP3R channels were also associated with alterations in mitochondrial calcium flux, especially in cardiomyocytes under HH exposure. IP3R-mediated calcium signaling required quantification of PACS2 and proximity of the ER and mitochondria. The supplement of PACS2 could improve mitochondrial respiration efficiency during HH exposure. Our previous randomized double-blinded clinical trial proposed that cardiac function could be recovered by optimizing myocardial energy metabolism. 54 Combined with those results, our present results provide a therapy for improving cardiac function at high altitudes targeted on energy metabolic reprogramming based on calcium flux across the MAM in cardiomyocytes. Moreover, linking reprogramming of energy metabolism induced by the PACS2 supplement was associated with enhanced mitophagy. We considered that mitophagy may, at least partly, provide relatively efficient substrates such as fatty acids for maintaining energy demand. However, the exact role of calcium in cardiomyocyte energy metabolism reprogramming requires confirmation using accurate techniques such as isotope tracing analysis.
In conclusion, we described acardiomyocyte injury mechanism during HH exposure in high-altitude environments. HH downregulated the expression of PACS2 in the MAM. Decreased PACS2 disrupted MAM formation and calcium transfer from the ER to the mitochondria, leading to mitophagy inhibition and mitochondrial energy metabolism impairment, which induced cardiomyocyte injury and right cardiac dysfunction during HH exposure. Our study identified a potential target for the prevention and treatment of cardiovascular diseases caused by high-altitude exposure.

Limitations of the study
Due to the limitations in the present study, we did not explore the possible molecular mechanisms that contribute to the downregulation of PACS2 in cardiomyocytes under HH conditions. Besides, MAM is also abundant and important in the left ventricular, 19,36 therefore further studies are warranted to investigate the different chamber's expression of PACS2 response to HH exposure and its role in modulating the cardiac function. Finally, there were no human samples from patients. The clinical relevance of the above findings needs further validation in the future.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

AUTHOR CONTRIBUTIONS
LH contributed to the conception and design of the research. JY, MS, and RC performed experiments and drafted the article. XY and BW helped with data acquisition and discussion. RC, CH, and ZL analyzed the data with guidance from JZ and XG. All authors read and approved the final submission of the article.

DECLARATION OF INTERESTS
The authors declare no competing interests. iScience Article Bioinformatics data analysis The UPLC-Q-TOF/MS raw data were analyzed using progenesis QI (Waters CorporationMilford, USA) software. The parameters used were retention time (RT) range 0.5-14.0 minutes, mass range 50-1000 Da, and mass tolerance 0.01 Da. Isotopic peaks were excluded from the analysis, noise elimination level was set at 10.00, minimum intensity was set to 15% of base peak intensity, and RT tolerance was set at 0.01 minute. The excel file was obtained with three-dimensional data sets including m/z, peak RT, and peak intensities; RT-m/z pairs were used as the identifier for each ion. The resulting matrix was further reduced by removing any peaks with missing values (ion intensity = 0) in > 60% of samples. The internal standard was used for data quality control (reproducibility). The positive and negative data were combined to yield a combined data set imported into SIMCA-P + 14.0 software package (Umetrics, Umeå , Sweden). Principle component analysis and (orthogonal) partial least-squares-discriminant analysis ([O] PLS-DA) were performed to visualize the metabolic alterations among the experimental groups, after mean centering and unit variance scaling. Tandem mass spectra were processed by PEAKS Studio version X (Bioinformatics Solutions Inc., Waterloo, Canada). Differentially expressed proteins were filtered if they contained R 1 unique peptide with P % 0.05 and fold change R 1.2. The pathway analysis was performed using GO and the KEGG database. iScience Article QUANTIFICATION AND STATISTICAL ANALYSIS All statistical analyses were performed with SPSS 20.0 software (Inc., USA). The measurement variables were presented as mean G standard deviation (SD) in minimum triplicates. Statistical significance was determined using Student's t-test between two groups and corrected for multiple comparisons (least-significant difference) for more than two groups. Mann-Whiney U test or nonparametric analysis of variance (Kruskal-Wallis) followed by Dunn's multiple comparison post-hoc test was used when one or more datasets showed non-normal distribution. Imaging experiments and animal tests were assessed in a blinded fashion. Sample sizes were similar between experimental groups and replicates of experiments. The number of biological replicates and observations are described in the figure legends. Statistical significance was considered at P < 0.05, with *P < 0.05, **P < 0.01. For graphs, all data were analyzed using GraphPad Prism software (version 8.4.0; GraphPad Software Inc, San Diego, CA).